Today I had to make some minor adjustments to an already established (and normally working pipeline). A big part of the pipeline is the filtering of cells transfected with HA-tag and untransfected cells.
I do this by filtering transfected cells through a minimum standard deviation value x of HAtag and a minimum median intensity value y of HAtag. For the untransfected cells I do the opposite, requiring the cells to have a maximum value of x and y for standard deviation and median intensity of HAtag.
This normally works fine, but now the pipeline won’t identify any untransfected cells, all while correctly identifying the transfected cells. I have attached the pipeline and an example image that should include 3 transfected cells and 2 untransfected cells.
I have tried everything I can think of, but nothing has worked so far. I could really use some help since I want to present this data on a lab meeting on Thursday this week.
NIH3T3 E40Q 20x 03-Image Export-17_b0c0x0-1388y0-1040.tif (4.1 MB) NIH3T3 E40Q 20x 03-Image Export-17_b0c1x0-1388y0-1040.tif (4.1 MB) NIH3T3 E40Q 20x 03-Image Export-17_b0c2x0-1388y0-1040.tif (4.1 MB) NIH3T3 E40Q 20x 03-Image Export-17_b0c0-3x0-1388y0-1040.tif (4.1 MB)
Side note: I also have a module that filters out “dead/damaged” cells with high intensities. One of this type of cells is filtered out earlier in the pipeline.
Thank you so much!